Profilin is the prototype of small proteins that sequester actin monomers. Recently a host of demonstrated or putative new functions for profilin have emerged. Genetic experiments on flies and yeast established a role for profilin in the function of the actin cytoskeleton. Biochemical studies suggest a dual role for profilin as a component of the phosphoinositide signal transduction machinery and as a messenger from the membrane to actin and other ligands. In addition, the ability to bind poly-L-proline is a common feature among profilins. This system is also ideal for testing the Structure-Based Thermodynamic Analysis (SBTA) model developed by Freire and others. This model predicts the thermodynamic parameters describing protein folding/unfolding using only the change in buried polar and apolar area as structure-specific information. The deltaH, delta S and deltaG for the binding of angiotensin II to an antibody were quantitatively predicted by this model. We plan to probe its range of validity by using it to analyze the binding of proline-rich peptides to profilin. We thus propose to: Refine and compare the NMR and x-ray structures of profilin I from Acanthamoeba. Compare the structures of Acanthamoeba and vertebrate profilin as a basis for phylogenetic analysis. Identify high affinity proline-rick peptides and proteins that bind to the polyproline binding site on profilin using a structure based fluorescent derivatives of profilin and phage display methods, determine the structure of the complex of these peptides with profilin by NMR or crystallography. Characterize the energetics of binding of profilin to polyproline and to the proline rich ligands; determine if the measured values are predicted by the SBTA model of murphy and Freire. Acanthamoeba actophorin is a member of a family of actin monomer binding/actin filament severing proteins that includes actin depolymerizing factor, destrin, cofilin, and depactin. The physiological functions and mechanisms of action of these proteins is still under investigations. To provide a structural basis for advanced studies of these proteins, we propose to: Complete and refine the actophorin crystal structure by the MAD phasing method. Crystalize and determine the structure of vertebrate cofilin for comparison with actophorin. Crystalize the complex of actophorin with SADP-actin. Explore the mechanism of actin-filament severing by modeling of the actin- actophorin complex. We will attempt to crystalize a number of actin- binding proteins.